This invention relates to Gxcex1q protein variants and their use in the analysis and discovery of agonists and antagonsists of chemosensory receptors, such as G protein coupled receptors involved in sensing of tastants, olfactants, and pheromones.
Heterotrimeric G proteins, consisting of alpha, beta and gamma subunits, couple ligand-bound seven transmembrane domain receptors (GPCRs or G-protein coupled receptors) to the regulation of effector proteins and production of intracellular second messengers such as cAMP, cGMP, and Ca2+. G protein signaling mediates the perception of environmental cues in all higher eukaryotic organisms, including yeast, Dictyostelium, plants, and animals. Agonist-bound sensory receptors catalyze the exchange of GTP for GDP on the surface of the Gx subunit to initiate intracellular responses to extracellular signals. Intracellular signaling is mediated through various effector enzymes, including cGMP phosphodiesterase, phospholipase C, adenylate cyclase, etc. (see Kinnamon and Margolskee, 1996, Curr. Opinion Neurobiol. 6: 506-513). Most effector proteins interact with the Gxcex1, although Gxcex2xcex3 subunits also contribute to the specificity of receptor-G protein coupling (Xu et al., 1998, J. Biol. Chem. 273(42): 27275-79).
The G protein xcex1 subunits are grouped into four families, Gxcex1s, Gxcex1i, Gxcex1q, and Gxcex112 according to their sequence homologies and functional similarities. The Gxcex1q family members couple a large group of GPCRs to phospholipase C. Activation of Gxcex1q coupled GPCRs induces intracellular calcium release and the capacitative calcium entry from extracellular space. The consequential increase of cytosolic calcium concentration can be effectively detected by using synthetic or genetically-engineered fluorescent calcium indicators, bioluminescent calcium indicators, calcium-activated ion currents, and by monitoring calcium-regulated gene transcription. Assays based on such calcium readout are available in high-throughput screening (HTS) format.
Signaling specificity among a subunits of the same class having similar biochemical functions is not well understood in vivo. For instance, the Gxcex1q (Gq) class includes four proteins expressed in mammals, called Gxcex1q, Gxcex111, Gxcex114, and Gxcex115 (in mice, Gxcex116 in humans). Whereas orthologs of these subunits are highly conserved across species (99, 97, 96 and 85% identity, respectively), paralogs of these subunits (expressed in the same species) are not as conserved. This suggests that each type of subunit in the Gq class has a distinct function, however, when transfected into Sf9 cells, the subunits stimulated phospholipase C with similar potency and showed similar activities (Nakamura et al., 1995, J. Biol. Chem. 270: 6246-6253). Xu and colleagues subsequently showed by gene knockouts in mice that Gqxcex1 subunits promiscuously couple to several different receptors in various cell types (1998, J. Biol. Chem. 273(42): 27275).
The promiscuity of the Gxcex1q subclass of G protein subunits provides a valuable tool for analyzing the role of G protein complexes and GPCRs in chemosensory transduction. For instance, the ability of Gxcex1q proteins to bypass the selectivity of the receptor G-protein interaction can be used to study the molecular mechanism of receptor-induced G-protein activation. In addition, the promiscuity toward receptors may be helpful in identifying ligands corresponding to orphan receptors whose signaling properties are unknown. Promiscuous G protein subunits play a particularly useful role in generating screening assays for high affinity GPCR agonists, antagonists, and modulators of chemosensory activity, in that using a single G protein coupler removes the variability of the G protein from the equation, thereby simplifying interpretation of results gleaned from various modulating compounds and GPCRs. Chemosensory modulating compounds involved in taste and/or smell, for instance, could then be used in the pharmaceutical and food industries to customize taste or aroma. In addition, such chemosensory molecules could be used to generate topographic maps that elucidate the relationship between the taste cells of the tongue or olfactory receptors (Ors) and sensory neurons leading to the brain.
Despite their promiscuity, however, Gxcex1q class subunits do not mediate all GPCRxe2x80x94effector interactions. For instance, human Gxcex116 and its murine counterpart Gxcex115 are promiscuous G proteins in that they couple to GPCRs of different G protein families (Offermanns and Simon, 1995; Negulescu et al., 1997). However, they are not true universal adapters for GPCRs in that there are at least 11 GPCRs reported to be incapable of activating Gxcex115/Gxcex116 (Wu et al., 1992; Arai et al., 1996; Kuang et al., 1996; Lee et al., 1998; Parmentier et al., 1998; Mody et al., 2000). Similar problems arise when using Gxcex115/xcex116 to identify ligands of ORs and T2Rs (bitter taste receptors) in that (1) calcium responses to odorants are small and quickly desensitized for ORs in Gxcex115/xcex116 transiently transfected cells (Krautwurst et al., 1998); (2) most T2Rs remain orphan using cell lines stably transfected with Gxcex115 (Adler et al., 2000; Chandrashekar et al., 2000); and (3) threshold concentration of denatonium measured is at least one order higher than expected for bitter receptors, hT2R4 and mT2R8 expressed in cells stably transfected with Gxcex115 (Adler et al., 2000; Chandrashekar et al., 2000). These problems suggest that the coupling efficiency between ORs/T2Rs and Gxcex115/xcex116 is weak and may vary within the family of ORs and T2Rs.
Given the partial promiscuity of Gxcex1q class proteins, it would be desirable to identify or create Gxcex1 protein subunits that are more promiscuous than their native counterparts, and which are capable of interacting with a wider variety GPCRs.
The present invention addresses the above described problems associated with using Gxcex115/xcex116, as well as other problems known in the art relating to the use of weakly promiscuous Gxcex1 proteins. Generally, the invention provides a series of Gxcex1q (Gq class) protein variants that functionally couple sensory cell receptors such as taste GPCRs (TRs) and olfactory GPCRs (ORs). According to the invention, the functional coupling can be determined, for example, by measuring changes in intracellular IP3 or calcium. In a particular embodiment, the Gq protein variants can be expressed in mammalian cell lines or Xenopus oocytes, and then evaluated using calcium fluorescence imaging and electrophysiological recording.
In one aspect of the invention, G alpha class q (Gq) variants that are capable of widely promiscuous functional coupling to chemosensory receptors, such as taste and olfactory receptors, and isolated nucleic acid sequences encoding the same are provided. Another aspect of the invention is directed to chimeric Gq variants and the isolated nucleic acids encoding the same. In one embodiment, the chimeric Gq protein variants comprise C-terminal sequences from transducin or Gxcex1olf, which exhibit improved functional coupling to taste and olfactory receptors, respectively.
In yet another aspect of the invention, a method for the analysis and discovery of agonists and/or antagonists of chemosensory receptors using the Gq protein variants is provided. One embodiment is directed to a mammalian cell-based assay using a transiently transfected gene or cDNA encoding a Gq protein variant. Another embodiment is directed to a mammalian cell-based assay using a stably expressed gene or cDNAs encoding Gq protein variants. In yet another embodiment, a method for analysis and discovery of agonists and/or antagonists of chemosensory receptors in Xenopus oocytes using genes, RNAs or DNAs encoding Gq protein variants is provided. The agonists and/or antagonists discovered using the disclosed assays are also encompassed, as are antibodies which bind specifically to the Gq variants described herein, but not those which also bind to known Gq proteins.
Other aspects of the invention relate to expression vectors comprising nucleic acid sequences encoding the Gq protein variants of the invention, as well as host cells comprising such expression vectors. Further aspects of the invention will become apparent to one of skill in the art from the following detailed description and examples.